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The nature of the immune responses associated with COVID-19 pathogenesis and disease severity, as well as the breadth of vaccine coverage and duration of immunity, is still unclear. Given the unpredictability for developing a severe/complicated disease, there is an urgent need in the field for predictive biomarkers of COVID-19. We have analyzed IgG Fc N-glycan traits of 82 SARS-CoV-2+ unvaccinated patients, at diagnosis, by nano-LC-ESI-MS. We determined the impact of IgG Fc glyco-variations in the induction of NK cells activation, further evaluating the association between IgG Fc N-glycans and disease severity/prognosis. We found that SARS-CoV-2+ individuals display, at diagnosis, variations in the glycans composition of circulating IgGs. Importantly, levels of galactose and sialic acid structures on IgGs are able to predict the development of a poor COVID-19 disease. Mechanistically, we demonstrated that a deficiency on galactose structures on IgG Fc in COVID-19 patients appears to induce NK cells activation associated with increased release of IFN-γ and TNF-α, which indicates the presence of pro-inflammatory immunoglobulins and higher immune activation, associated with a poor disease course. This study brings to light a novel blood biomarker based on IgG Fc glycome composition with capacity to stratify patients at diagnosis.
Assuntos
COVID-19 , Biomarcadores , COVID-19/diagnóstico , Teste para COVID-19 , Galactose , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Polissacarídeos , SARS-CoV-2 , Índice de Gravidade de DoençaRESUMO
Miscibility is an important issue in biopolymer blends for analysis of the behavior of polymer pairs through the detection of phase separation and improvement of the mechanical and physical properties of the blend. This study presents the formulation of a stable and one-phase mixture of collagen and regenerated silk fibroin (RSF), with the highest miscibility ratio between these two macromolecules, through inducing electrostatic interactions, using salt ions. For this aim, a ternary phase diagram was experimentally built for the mixtures, based on observations of phase behavior of blend solutions with various ratios. The miscibility behavior of the blend solutions in the miscible zones of the phase diagram was confirmed quantitatively by viscosimetric measurements. Assessing the effects of biopolymer mixing ratio and salt ions, before and after dialysis of blend solutions, revealed the importance of ion-specific interactions in the formation of coacervate-based materials containing collagen and RSF blends that can be used in pharmaceutical, drug delivery, and biomedical applications. Moreover, the conformational change of silk fibroin from random coil to beta sheet, in solution and in the final solid films, was detected by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR), respectively. Scanning electron microscopy (SEM) exhibited alterations of surface morphology for the biocomposite films with different ratios. Surface contact angle measurement illustrated different hydrophobic properties for the blended film surfaces. Differential scanning calorimetry (DSC) showed that the formation of the beta sheet structure of silk fibroin enhances the thermal stability of the final blend films. Therefore, the novel method presented in this study resulted in the formation of biocomposite films whose physico-chemical properties can be tuned by silk fibroin conformational changes by applying different component mixing ratios.
Assuntos
Biopolímeros/química , Colágeno/química , Fibroínas/química , Seda/química , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Biopolímeros/biossíntese , Bombyx/química , Microscopia Eletrônica de Varredura , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa). The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition. We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/toxicidade , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dicroísmo Circular , Relação Dose-Resposta a Droga , Membrana Eritrocítica/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipídeos de Membrana/química , Membranas Artificiais , Testes de Sensibilidade Microbiana , Oligopeptídeos/química , Oligopeptídeos/toxicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Relação Estrutura-AtividadeRESUMO
AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39)-Glu(40) peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Toxinas Bacterianas/metabolismo , Metaloproteases/metabolismo , Photobacterium/metabolismo , Fator de Transcrição RelA/metabolismo , Fatores de Virulência/metabolismo , Animais , Bass , Doenças dos Peixes/metabolismo , Interações Hospedeiro-Patógeno , Leucócitos/metabolismo , Leucócitos/patologia , Proteínas RecombinantesRESUMO
Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/metabolismo , Dicroísmo Circular , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx(-)) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx(-) was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx(-) were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47(phox-/-) and B6.RAG2(-/-) IFN-γ(-/-) mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx(-). A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx(-) were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity.
Assuntos
Leishmania/enzimologia , Leishmaniose/enzimologia , Mitocôndrias/enzimologia , Peroxirredoxinas/metabolismo , Animais , Células Cultivadas , Citrato (si)-Sintase/metabolismo , Modelos Animais de Doenças , Interações Hospedeiro-Parasita , Leishmania/crescimento & desenvolvimento , Leishmania/patogenicidade , Leishmaniose/imunologia , Leishmaniose/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/parasitologia , Chaperonas Moleculares , Carga ParasitáriaRESUMO
Existent research shows that affective polarization has been intensifying in some publics, diminishing in others, and remaining stable in most. We contribute to this debate by providing the most encompassing comparative and longitudinal account of affective polarization so far. We resort to a newly assembled dataset able to track partisan affect, with varying time series, in eighteen democracies over the last six decades. We present results based on two different operational measures of affective polarization: Reiljan's Affective Polarization Index, based on reported partisans only, and Wagner's weighted distance from the most liked party, based on the whole electorate. Our reassessment of affective polarization among partisans confirms that an intensifying trend is observable in a number of countries but it is, by no means, generalizable to all established democracies. Regarding the longitudinal assessment of affective polarization among the electorate, we confirm that US citizens have become more affectively polarized over time.
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This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems.
Assuntos
Bases de Dados de Produtos Farmacêuticos , Ligantes , Proteínas Recombinantes/genética , Expressão Gênica/genéticaRESUMO
The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor involved in various human diseases, as it binds to numerous molecules and proteins that modulate the activity of other proteins. Elucidating the three-dimensional structure of this receptor is therefore most important for understanding its function during activation and cellular signaling. The major alternative splice product of RAGE comprises its extracellular region that occurs as a soluble protein (sRAGE). Although the structures of sRAGE domains were available, their assembly into the functional full-length protein remained unknown. We observed that the protein has concentration-dependent oligomerization behavior, and this is also mediated by the presence of Ca(2+) ions. Moreover, using synchrotron small angle x-ray scattering, the solution structure of human sRAGE was determined in the monomeric and dimeric forms. The model for the monomer displays a J-like shape, whereas the dimer is formed through the association of the two N-terminal domains and has an elongated structure. These results provide insights into the assembly of the RAGE homodimer, which is essential for signal transduction, and the sRAGE:RAGE heterodimer that leads to blockage of the receptor signaling, paving the way for the design of therapeutic strategies for a large number of different pathologies.
Assuntos
Cálcio/química , Multimerização Proteica/fisiologia , Receptores Imunológicos/química , Cálcio/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/fisiologia , Solubilidade , Relação Estrutura-AtividadeRESUMO
The remarkable properties of poly-aminoacids, mainly their biocompatibility and biodegradability, have prompted an increasing interest in these polymers for biomedical applications. Poly-γ-glutamic acid (γ-PGA) is one of the most interesting poly-aminoacids with potential applications as a biomaterial. Here we describe the production and characterization of γ-PGA by Bacillus subtilis natto. The γ-PGA was produced with low molecular weight (10-50 kDa), high purity grade (>99 %) and a D: -/L: -glutamate ratio of 50-60/50-40 %. To evaluate the feasibility of using this γ-PGA as a biomaterial, chitosan (Ch)/γ-PGA nanoparticles were prepared by the coacervation method at pH ranging from 3.0 to 5.0, with dimensions in the interval 214-221 nm with a poly-dispersion index of ca. 0.2. The high purity of γ-PGA produced by this method, which is firstly described here, renders this biopolymer suitable for biomedical applications. Moreover, the Ch/γ-PGA nanocomplexes developed in this investigation can be combined with biologically active substances for their delivery in the organism. The fact that the assembly between Ch and γ-PGA relies on electrostatic interactions enables addition of other molecules that can be released into the medium through changes from acidic to physiological pH, without loss in biological activity.
Assuntos
Materiais Biocompatíveis , Ácido Poliglutâmico/análogos & derivados , Bacillus subtilis/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , EstereoisomerismoRESUMO
Recent developments in Western societies have motivated a growing consideration of the role of negativity in public opinion and political behavior research. In this article, we review the scant (and largely disconnected) scientific literature on negativity and political behavior, merging contributions from social psychology, public opinion, and electoral research, with a view on developing an integrated theoretical framework for the study of negative voting in contemporary democracies. We highlight that the tendency toward negative voting is driven by three partly overlapping components, namely, (1) an instrumental-rational component characterized by retrospective performance evaluations and rationalization mechanisms, (2) an ideological component grounded on long-lasting political identities, and (3) an affective component, motivated by (negative) attitudes toward parties and candidates. By blueprinting the systematic relationships between negative voting and each of these components in turn, and suggesting multiple research paths, this article aims to stimulate future studies on negative voting in multi-party parliamentary systems to motivate a better understanding of the implications of negativity in voting behavior in contemporary democracies.
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About one third of American voters cast a vote more "against" than "for" a candidate in the 2020 Presidential election. This pattern, designated by negative voting, has been initially understood by rational choice scholarship as a product of cognitive dissonance and/or retrospective evaluations. This article revisits this concept through the affective polarization framework in the light of the rise of political sectarianism in American society. Based on an original CAWI survey fielded after the 2020 election, our regression analysis demonstrates that the predicted probability of casting a negative vote significantly increases among individuals for whom out-candidate hate outweighs in-candidate love. Negative voting is less prevalent among partisans as their higher levels of in-group affection can offset out-group contempt. By asserting the enduring relevance of negative voting in American presidential elections, we aim at stimulating further research and discussion of its implications for democratic representation.
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A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4(T) has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal.
Assuntos
Actinomycetales/enzimologia , Amidoidrolases/metabolismo , Azepinas/metabolismo , Proteínas de Bactérias/metabolismo , Cobalto/metabolismo , Herbicidas/metabolismo , Tiocarbamatos/metabolismo , Actinomycetales/classificação , Actinomycetales/genética , Actinomycetales/metabolismo , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
Chitosan (Ch) is a nontoxic and biocompatible polysaccharide extensively used in biomedical applications. Ch, as a polycation, can be combined with anionic polymers by layer-by-layer (LbL) self-assembly, giving rise to multilayered complexed architectures. These structures can be used in tissue engineering strategies, as drug delivery systems, or artificial matrices mimicking the extracellular microenvironment. In this work, Ch was combined with poly(γ-glutamic acid) (γ-PGA). γ-PGA is a polyanion, which was microbially produced, and is known for its low immunogenic reaction and low cytotoxicity. Multilayered ultrathin films were assembled by LbL, with a maximum of six layers. The interaction between both polymers was analyzed by: ellipsometry, quartz crystal microbalance with dissipation, Fourier transform infrared spectroscopy, atomic force microscopy, and zeta potential measurements. Ch/γ-PGA polyelectrolyte multilayers (PEMs) revealed no cytotoxicity according to ISO 10993-5. Overall, this study demonstrates that Ch can interact electrostatically with γ-PGA forming multilayered films. Furthermore, this study provides a comprehensive characterization of Ch/γ-PGA PEM structures, elucidating the contribution of each layer for the nanostructured films. These model surfaces can be useful substrates to study cell-biomaterial interactions in tissue regeneration.
Assuntos
Quitosana/metabolismo , Eletrólitos/síntese química , Ácido Poliglutâmico/análogos & derivados , Materiais Biocompatíveis/química , Quitosana/análise , Quitosana/química , Sistemas de Liberação de Medicamentos/métodos , Eletrólitos/análise , Eletrólitos/química , Microscopia de Força Atômica , Modelos Moleculares , Ácido Poliglutâmico/análise , Ácido Poliglutâmico/biossíntese , Ácido Poliglutâmico/metabolismo , Engenharia Tecidual/métodos , Substâncias Viscoelásticas/análise , Substâncias Viscoelásticas/químicaRESUMO
The Picornaviridae family contains a large number of human pathogens such as rhinovirus, poliovirus and hepatitis A virus (HAV). Hepatitis A is an infectious disease that causes liver inflammation. It is highly endemic in developing countries with poor sanitation, where infections often occur in children. As in other picornaviruses, the genome of HAV contains one open reading frame encoding a single polyprotein that is subsequently processed by viral proteinases to originate mature viral proteins during and after the translation process. In the polyprotein, the N-terminal P1 region generates the four capsid proteins, while the C-terminal P2 and P3 regions contain the enzymes, precursors and accessory proteins essential for polyprotein processing and virus replication. Here, the first crystals of protein 2AB of HAV are reported. The crystals belonged to space group P4(1) or P4(3), with unit-cell parameters a = b = 90.42, c = 73.43 Å, and contained two molecules in the asymmetric unit. Native and selenomethionine-derivative crystals diffracted to 2.7 and 3.2 Å resolution, respectively.
Assuntos
Vírus da Hepatite A/enzimologia , Peptídeo Hidrolases/química , Clonagem Molecular , Cristalografia por Raios X , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificaçãoRESUMO
Grape pectic polysaccharides-malvidin-3-O- ß -d-glucoside binding was studied, aiming to unveil the impact of structural diversity of polysaccharides on anthocyanins-polysaccharides interactions. Polysaccharides were extracted with solutions of imidazole (ISP) and carbonate at 4 °C (CSP-4 °C) and room temperature (CSP-RT) and also recovered from the dialysis supernatant of the remaining cellulosic residue after the aqueous NAOH extraction of hemicellulosic polysaccharides (Sn-CR). Polysaccharides richer in homogalacturonan domains, like those present in the CSP-4 °C fraction had approximately 50-fold higher binding affinity to malvidin-3-O- ß-d-glucoside, than polysaccharides with side chains (as ISP and CSP-RT extractable polysaccharides). CSP-4 °C polysaccharides showed a positive effect on malvidin-3-O- ß-d-glucoside colour fading. Hydration equilibrium constant of malvidin-3-O- ß-d-glucoside in the presence of CSP-4 °C polysaccharides was higher, showing the preferential stabilization of the flavylium cation. The results showed that anthocyanins colour stabilization can be promoted by pectic polysaccharide structures such as those extracted by cold carbonate.
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BACKGROUND: Arabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL. RESULTS: The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein. CONCLUSIONS: The Arabidopsis thaliana transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.
Assuntos
Arabidopsis/genética , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Clonagem Molecular , Proteínas de Membrana/genética , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The cultivation of genetically modified organisms (GMO) continues to expand worldwide. Still, many consumers express concerns about the use of GMO in food or feed, and many countries have legislated on labelling systems to indicate the presence of GMO in commercial products. To deal with the increased number of GMO events and to address related regulations, alternative detection methods for GMO inspection are required. In this work, a genosensor based on Surface Plasmon Resonance under continuous flow was developed for the detection and quantification of a genetically modified soybean (event GTS 40-3-2). In a single chip, the simultaneous detection of the event-specific and the taxon-specific samples were achieved, whose detection limits were 20 pM and 16 pM, respectively. The reproducibility was 1.4%, which supports the use of the chip as a reliable and cost-effective alternative to other DNA-based techniques. The results indicate that the proposed method is a versatile tool for GMO quantification in food and feed samples.
Assuntos
Glycine max/genética , Ressonância de Plasmônio de Superfície/métodos , DNA de Plantas/genética , Alimentos Geneticamente Modificados/classificação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Organismos Geneticamente Modificados/química , Organismos Geneticamente Modificados/genética , Plantas Geneticamente Modificadas/genética , Reprodutibilidade dos TestesRESUMO
α-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45⯰C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease.
Assuntos
Lipídeos de Membrana/química , Polímeros/farmacologia , Lipossomas Unilamelares/química , alfa-Sinucleína/química , Alcenos/química , Alcenos/farmacologia , Humanos , Proteínas Intrinsicamente Desordenadas/química , Bicamadas Lipídicas/química , Maleatos/química , Maleatos/farmacologia , Proteínas de Membrana/química , Polímeros/química , Agregados Proteicos/efeitos dos fármacos , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacosRESUMO
The first contact of tannins with the human body occurs in the mouth, where some of these tannins are known to interact with salivary proteins, in particular with proline-rich proteins (PRPs). These interactions are important at a sensory level, especially for astringency development, but could also affect the biological activities of the tannins. This study gathers information on the relative affinity of the interaction, complex stoichiometry, and tannin molecular epitopes of binding for the interactions between the families of PRPs (bPRPs, gPRPs, and aPRPs) and three representative ellagitannins (castalagin, vescalagin, and punicalagin). These interactions were studied by saturation-tranfer difference NMR and microcalorimetry. The effect of the PRP-ellagitannin interaction on their antioxidant ability was also assessed by ferric reduction antioxidant power (FRAP) assays. The results support a significant interaction between the studied tannins and PRPs with binding affinities in the micromolar range. Punicalagin was always the ellagitannin with higher affinity. aPRPs were the salivary PRPs with higher affinity. Moreover, it was observed that when ellagitannins are present in low concentrations (5-50 µM), as occurs in food, the antioxidant ability of these tannins when complexed with salivary PRPs could be significantly impaired.